Serious infections with category B select agents or other bacteria require urgent treatment. We propose to develop a user-friendly assay system that is appropriate for point-of-care detection of category B select agents and other common pathogenic bacteria. The system will integrate a hands-free method to process clinical blood samples for PCR with an exceptionally sensitive nested, real-time PCR assay capable of simultaneously detecting all category B bacterial select agents and other common medical pathogens in a single-step procedure. This system will offer unparalleled sensitivity by extracting bacterial DNA from all three "compartments" in whole blood: bacteria free in the blood, intracellular bacteria (principally in WBC) and free bacterial DNA, while simultaneously eliminating blood-associated human genomic DNA, which is an important source of PCR inhibition. Sample processing and automated nested PCR will be performed with a simple mini-fluidic cartridge prepackaged with all necessary reagents. Multiple pathogen detection will be performed using a multi-plex set of novel "sloppy" molecular beacons, which will generate precise "melting temperature codes" that are unique to each bacterial species. This application benefits from the substantial experience in molecular diagnostics and manipulation of select agents of investigators at New Jersey Medical School and the experience at cartridge development, sample processing and real-time PCR analysis at Cepheid Inc. The application is divided into four aims: (1) "assay development " will develop sloppy molecular beacon assays that can identify virtually all pathogenic bacteria in a single PCR assay;(2) Cartridge and reagent development" will develop a new "cartridge B" that extracts free and cell-associated bacterial DNA directly from blood, simultaneously separating and discarding human genomic DNA, and performs nested PCR in an integrated cartridge;(3) "software development" will develop software for automatic assay performance including data interpretation;(4) "optimization and design verification" will test the prototype system for sensitivity, specificity, limits of detection and asses the effects of potential interfering endogenous substances. Relevance of this research to public health: This application will make it possible to rapidly identify potentially life-threatening blood stream infections caused by category B bioterrorism agents or other bacteria, leading to improved diagnosis and treatment of these infections.